The present invention relates to oligonucleotides that have both A-form and B-form conformational geometry and methods of using such oligonucleotides. The oligonucleotides of the invention are useful in therapeutic and investigative purposes. More specifically, the present invention is directed to oligonucleotides having particular modifications that will increase affinity and nuclease resistance while concurrently serving as substrates for RNase H when bound to a target RNA strand.
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Classical therapeutic modes have generally focused on interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. However, recently, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, maximum therapeutic effect and minimal side effects may be realized. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides. Oligonucleotides are now accepted as therapeutic agents. A first such oligonucleotide has been approved for human therapeutic use by the FDA and is available in commercial marketplace.
Oligonucleotides are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another. The concept of inhibiting gene expression through the use of sequence-specific binding of oligonucleotides to target RNA sequences, also known as antisense inhibition, has been demonstrated in a variety of systems, including living cells (for example see: Wagner et al., Science (1993) 260: 1510-1513; Milligan et al., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem. Reviews, (1990) 90:543-584; Stein et al., Cancer Res., (1988) 48:2659-2668).
The events that provide the disruption of the nucleic acid function by antisense oligonucleotides (Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts""O, P. O. P. (1987) Anti-Cancer Drug Design, 2:117-128, and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are probably the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Oligonucleotides may also bind to duplex nucleic acids to form triplex complexes in a sequence specific manner via Hoogsteen base pairing (Beal et al., Science, (1991) 251:1360-1363; Young et al., Proc. Natl. Acad. Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeutic strategies are directed towards nucleic acid sequences that are involved in or responsible for establishing or maintaining disease conditions. Such target nucleic acid sequences may be found in the genomes of pathogenic organisms including bacteria, yeasts, fungi, protozoa, parasites, viruses, or may be endogenous in nature. By hybridizing to and modifying the expression of a gene important for the establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides may also be of therapeutic value when they bind to non-nucleic acid biomolecules such as intracellular or extracellular polypeptides, proteins, or enzymes. Such oligonucleotides are often referred to as xe2x80x98aptamersxe2x80x99 and they typically bind to and interfere with the function of protein targets (Griffin, et al., Blood, (1993), 81:3271-3276; Bock, et al., Nature, (1992) 355: 564-566).
Oligonucleotides and their analogs have been developed and used for diagnostic purposes, therapeutic applications and as research reagents. For use as therapeutics, oligonucleotides must be transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These critical functions depend on the initial stability of the oligonucleotides toward nuclease degradation. A serious deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is the enzymatic degradation of administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes referred to as nucleases. For oligonucleotides to be useful as therapeutics or diagnostics, the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation. For a non-cellular use such as a research reagent, oligonucleotides need not necessarily possess nuclease stability.
A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation.
Modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and the use of modified sugar moieties such as 2xe2x80x2-O-alkyl ribose. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. A number of modifications that dramatically alter the nature of the internucleotide linkage have also been reported in the literature. These include non-phosphorus linkages, peptide nucleic acids (PNA""s) and 2xe2x80x2-5xe2x80x2 linkages. Another modification to oligonucleotides, usually for diagnostic and research applications, is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
A variety of modified phosphorus-containing linkages have been studied as replacements for the natural, readily cleaved phosphodiester linkage in oligonucleotides. In general, most of them (such as the phosphorothioate, phosphoramidates, phosphonates and phosphorodithioates) result in oligonucleotides with reduced binding to complementary targets and decreased hybrid stability. At least one dozen phosphorothioate oligonucleotides and derivatives are presently being used as antisense agents in human clinical trials for the treatment of various disease states. The antisense drug Vitravine(trademark), for use to treat cytomegalovirus (CMV) retinitis in humans, has been approved by regulatory agencies and is comedically marketed.
The structure and stability of chemically modified nucleic acids is of great importance to the design of antisense oligonucleotides. Over the last ten years, a variety of synthetic modifications have been proposed to increase nuclease resistance, or to enhance the affinity of the antisense strand for its target mRNA (Crooke et al., Med. Res. Rev., 1996, 16,319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374).
RNA exists in what has been termed xe2x80x9cA Formxe2x80x9d geometry, while DNA exists in xe2x80x9cB Formxe2x80x9d geometry. In general, RNA:RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997,25,2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of a hydroxyl group in the 2xe2x80x2-pentofuranosyl (i.e., 2xe2x80x2-sugar) position in RNA is believed to bias the sugar toward a C3xe2x80x2 endo pucker (also known as a Northern pucker), which causes the duplex to favor the A-form geometry. On the other hand, 2xe2x80x2-deoxy nucleic acids (those having 2xe2x80x2-deoxy-erythro-pentofuranosyl nucleotides) prefer a C2xe2x80x2 endo sugar pucker (also known as Southern pucker), which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). In addition, the 2xe2x80x2 hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494).
DNA:RNA hybrid duplexes are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of a DNA:RNA hybrid is central to antisense therapies as the mechanism requires the binding of a modified DNA strand to a mRNA strand. To effectively inhibit the mRNA, the antisense DNA should have a very high binding affinity with the mRNA. Otherwise the desired interaction between the DNA and target mRNA strand will occur infrequently, thereby decreasing the efficacy of the antisense oligonucleotide.
One synthetic 2xe2x80x2-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2xe2x80x2-methoxyethoxy (MOE, 2xe2x80x2-OCH2CH2OCH3) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443). One of the immediate advantages of the MOE substitution is the improvement in binding affinity, which is greater than many similar 2xe2x80x2 modifications such as O-methyl, O-propyl, and O-aminopropyl (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443). 2xe2x80x2-O-Methoxyethyl-substituted compounds also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Such compounds typically display improved RNA affinity and higher nuclease resistance relative to DNA. Chimeric oligonucleotides with 2xe2x80x2-O-methoxyethyl-ribonucleoside wings and a central DNA-phosphorothioate window also have been shown to effectively reduce the growth of tumors in animal models at low doses. MOE substituted oligonucleotides have shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.
Recently Damha et. al., published two paper describing certain oligonucleotides that utilized arabino-pentofuranosyl nucleotides as building blocks (Damha et. al., J.A.C.S., 1998, 120, 12976-12977 and Damha et. al., Bioconjugate Chem., 1999, 10, 299-305). The arabino-pentofuranosyl oligonucleotides, i.e., arabinonucleic acids, described by Damha et. al., utilized either arabinose or 2xe2x80x2-deoxy-2xe2x80x2-fluoro arabinose as the sugar unit of their respective nucleotides. In one of the two arabinonucleic acids described, all of the nucleotides of the nucleic acid were arabinose and in the other, all of the nucleotides were 2xe2x80x2-deoxy-2xe2x80x2-fluoro arabinose. In both of these nucleic acids, the nucleotides were joined via phosphodiester linkages. These authors were able to show that the 2xe2x80x2-fluoro arabino-containing oligonucleotides when bound to RNA activated cleavage of the RNA by E. coli and HIV-RT RNase H. The authors further noted that while the two arabinonucleic acids they described were more stable to serum and cellular nucleases than DNA they were less stable than normal phosphorothioate deoxyoligonucleotides.
Although the known modifications to oligonucleotides have contributed to the development of oligonuclotides for various uses, including use in diagnostics, therapeutics and as research reagents, there still exists a need in the art for further oligonucleotides having enhanced hybrid binding affinity and/or increased nuclease resistance and that can take advantage of the RNase H termination mechanism.
In one aspect, the present invention is directed to oligonucleotides having multiple properties. One of these properties is the ability to form a double stranded structure with an RNA and elicit RNase H cleavage of the RNA. Further properties of the oligonucleotides include having improved binding affinity and nuclease resistance. The oligonucleotides of the invention comprise oligonucleotide formed from a plurality of nucleotides. A first portion of the nucleotides are joined together in a contiguous sequence with each nucleotide of this portion selected as a nucleotide that has B-form conformational geometry when joined in a contiguous sequence with other nucleotides. Included in this first portion of nucleotides are ribonucleotides or arabino nucleotides. The oligonucleotides include a further portion of nucleotides that are joined together in at least one contiguous sequence. Each of these further nucleotides are selected as ribonucleotides that have A-form conformational geometry when joined in a contiguous sequence.
In preferred embodiments of the invention, each of the nucleotides of the first portion of nucleotides, independently, are selected to be 2xe2x80x2-SCH3 ribonucleotides, 2xe2x80x2-NH2 ribonucleotides, 2xe2x80x2-NH(C1-C2 alkyl) ribonucleotides, 2xe2x80x2-N(C1-C2 alkyl)2 ribonucleotides, 2xe2x80x2-CF3 ribonucleotides, 2xe2x80x2=CH2 ribonucleotides, 2xe2x80x2=CHF ribonucleotides, 2xe2x80x2=CF2 ribonucleotides, 2xe2x80x2-CH3 ribonucleotides, 2xe2x80x2-C2H5 ribonucleotides, 2xe2x80x2-CHxe2x95x90CH2 ribonucleotides or 2xe2x80x2-Cxe2x89xa1CH ribonucleotides. These are joined together in a contiguous sequence by phosphate, phosphorothioate, phosphorodithioate or boranophosphate linkages.
In a further preferred embodiment of the invention, each of the nucleotides of said further portion of nucleotides, independently, are selected to be 2xe2x80x2-fluoro nucleotides or nucleotides having a 2xe2x80x2-substituent having the formula I or II: 
wherein
E is C1-C10 alkyl, N(Q1)(Q2) or Nxe2x95x90C(Q1)(Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support, or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
R3 is OX, SX, or N(X)2;
each X is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)Z, C(xe2x95x90O)N(H)Z or OC(xe2x95x90O)N(H)Z;
Z is H or C1-C8 alkyl;
L1, L2 and L3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1)(Q2), O(Q1), halo, S(Q1), or CN;
each q1 is, independently, from 2 to 10;
each q2 is, independently, 0 or 1;
m is 0, 1 or 2;
p is from 1 to 10; and
q3 is from 1 to 10 with the proviso that when p is 0, q3 is greater than 1.
A more preferred group for use as the further portion of nucleotides are 2xe2x80x2-F ribonucleotides, 2xe2x80x2-Oxe2x80x94(C1-C6 alkyl) ribonucleotides, or 2xe2x80x2-Oxe2x80x94(C1-C6 substituted alkyl) ribonucleotides wherein the substitution is C1-C6 ether, C1-C6 thioether, amino, amino(C1-C6 alkyl) or amino(C1-C6 alkyl)2. These nucleotides are joined together in sequence by 3xe2x80x2-5xe2x80x2 phosphodiester, 2xe2x80x2-5xe2x80x2 phosphodiester, phosphorothioate, Sp phosphorothioate, Rp phosphorothioate, phosphorodithioate, 3xe2x80x2-deoxy-3xe2x80x2-amino phosphoroamidate, 3xe2x80x2-methylenephosphonate, methylene(methylimino), dimethylhydazino, amide 3 (i.e., (3xe2x80x2)xe2x80x94CH2xe2x80x94NHxe2x80x94C(O)xe2x80x94(5xe2x80x2)), amide 4 (i.e., (3xe2x80x2)xe2x80x94CH2xe2x80x94C(O)xe2x80x94NHxe2x80x94(5xe2x80x2)) or boranophosphate linkages.
In one preferred embodiment of the invention, at least two of the nucleotides of the further portion of nucleotides are joined together in a contiguous sequence that is position 3xe2x80x2 to the contiguous sequence of the first portion of nucleotides. In an additional preferred embodiment of the invention, at least two of the further portion of nucleotides are joined together in a continuous sequence that is position 5xe2x80x2 to the continuous sequence of the first portion of nucleotides.
In a further preferred embodiment of the invention, at least two of the nucleotides of the further portion of nucleotides are joined together in a continuous sequence that is position 3xe2x80x2 to the continuous sequence of the first portion of nucleotides and at least two of the further portion of nucleotides are joined together in a continuous sequence that is position 5xe2x80x2 to the continuous sequence of the first portion of nucleotides.
A first preferred group of nucleotides for use as the first portion of nucleotides include 2xe2x80x2-SCH3 ribonucleotides, 2xe2x80x2-NH2 ribonucleotides, 2xe2x80x2-NH(C C2 alkyl) ribonucleotides, 2xe2x80x2-N(C1-C2 alkyl)2 ribonucleotides, 2xe2x80x2=CH2 ribonucleotides, 2xe2x80x2-CH3 ribonucleotides, 2xe2x80x2-C2H5 ribonucleotides, 2xe2x80x2-CHxe2x95x90CH2 ribonucleotides and 2xe2x80x2-Cxe2x89xa1CH ribonucleotides. A more preferred group include 2xe2x80x2-SCH3 ribonucleotides, 2xe2x80x2-NH2 ribonucleotides, 2xe2x80x2-NH(C1-C2 alkyl) ribonucleotides, 2xe2x80x2-N(C1-C2 alkyl)2 ribonucleotides and 2xe2x80x2-CH3 ribonucleotides. A further preferred group include 2xe2x80x2-SCH3 ribonucleotides, 2xe2x80x2-NH2 ribonucleotides and 2xe2x80x2-CH3 ribonucleotides. Particularly preferred are is 2xe2x80x2-SCH3 ribonucleotides.
A further group of nucleotides that are preferred of use as the nucleotides of the first portion of the oligonucleotides of the inventions are 2xe2x80x2-CN arabino nucleotides, 2xe2x80x2-F arabino nucleotides, 2xe2x80x2-Cl arabino nucleotides, 2xe2x80x2-Br arabino nucleotides, 2xe2x80x2-N3 arabino nucleotides, 2xe2x80x2-OH arabino nucleotides, 2xe2x80x2-Oxe2x80x94CH3 arabino nucleotides and 2xe2x80x2-dehydro-2xe2x80x2-CH3 arabino nucleotides. A more preferred group include 2xe2x80x2-F arabino nucleotides, 2xe2x80x2-OH arabino nucleotides and 2xe2x80x2-Oxe2x80x94CH3 arabino nucleotides. A further preferred group include 2xe2x80x2-F arabino nucleotides and 2xe2x80x2-OH arabino nucleotides. Particularly preferred are 2xe2x80x2-F arabino nucleotides.
Particularly preferred oligonucleotides of the invention include selecting the nucleotides of the first portion of nucleotides to be 2xe2x80x2-SCH3 ribonucleotides, 2xe2x80x2-NH2 ribonucleotides, 2xe2x80x2-NH(C1-C2 alkyl) ribonucleotides, 2xe2x80x2-N(C1-C2 alkyl)2 ribonucleotides, 2xe2x80x2-CH3 ribonucleotides, 2xe2x80x2-CHxe2x95x90CH2 ribonucleotides or 2xe2x80x2-Cxe2x89xa1CH ribonucleotides and selecting the nucleotides of the further portion of nucleotides to be 2xe2x80x2-F ribonucleotides, 2xe2x80x2-Oxe2x80x94(C1-C6 alkyl) ribonucleotides or 2xe2x80x2-Oxe2x80x94(C1-C6 substituted alkyl) ribonucleotides wherein the substitution is C1-C6 ether, C1-C6 thioether, amino, amino(C1-C6 alkyl) or amino(C1-C6 alkyl)2.
Further preferred oligonucleotides of the invention include selecting the nucleotides of said first portion of nucleotides to be 2xe2x80x2-CN arabino nucleotides, 2xe2x80x2-F arabino nucleotides, 2xe2x80x2-Cl arabino nucleotides, 2xe2x80x2-Br arabino nucleotides, 2xe2x80x2-N3 arabino nucleotides, 2xe2x80x2-OH arabino nucleotides, 2xe2x80x2-Oxe2x80x94CH3 arabino nucleotides or 2xe2x80x2-dehydro-2xe2x80x2-CH3 arabino nucleotides and selecting the nucleotides of the further portion of nucleotides to be 2xe2x80x2-F ribonucleotides, 2xe2x80x2-Oxe2x80x94(C1-C6 alkyl) ribonucleotides or 2xe2x80x2-Oxe2x80x94(C1-C6 substituted alkyl) ribonucleotides wherein the substitution is C1-C6 ether, C1-C6 thioether, amino, amino(C1-C6 alkyl) or amino(C1-C6 alkyl)2.
Particularly preferred are oligonucleotide of the invention where each nucleotide of the first portion of nucleotides is a 2xe2x80x2-F arabino nucleotides or a 2xe2x80x2-OH arabino nucleotides and each nucleotide of the further portion of nucleotides is a 2xe2x80x2-Oxe2x80x94(C1-C6 substituted alkyl) ribonucleotide wherein the substitution is C1-C6 ether, C1-C6 thioether, amino, amino(C1-C6 alkyl) or amino(C1-C6 alkyl)2.
In further preferred oligonucleotides of the invention the further portion of the plurality of nucleotides comprise at least two nucleotides joined together in a contiguous sequence that is position at the 3xe2x80x2 terminus end of the oligonucleotide. In an additional preferred oligonucleotide of the invention the further portion of said plurality of nucleotides comprise at least two nucleotides joined together in a contiguous sequence that is position at the 5xe2x80x2 terminus end of the oligonucleotide. In even further preferred oligonucleotides of the invention the further portion of the plurality of nucleotides comprise at least two nucleotides joined together in a contiguous sequence that is position at the 3xe2x80x2 terminus end of the oligonucleotide and at least two nucleotides joined together in a contiguous sequence that is position at the 5xe2x80x2 terminus end of the oligonucleotide. Preferred linkages for joining these nucleotides together in an oligonucleotide of the invention include 2xe2x80x2-5xe2x80x2 phosphodiester linkages, 3xe2x80x2-methylenephosphonate linkages, Sp phosphorothioate linkages, methylene(methylimino)linkages, dimethyhydrazino linkages, 3xe2x80x2-deoxy-3xe2x80x2-amino phosphoroamidate linkages, amide 3 linkages or amide 4 linkages. Particularly preferred joining linkages are 2xe2x80x2-5xe2x80x2 phosphodiester linkages, 3xe2x80x2-methylenephosphonate linkages, Sp phosphorothioate linkages or methylene(methylimino) linkages.
In further preferred oligonucleotides of the invention, nucleotides for use in the further portion of nucleotides comprises 2xe2x80x2-alkylamino substituted nucleotides located at the 3xe2x80x2 terminus, the 5xe2x80x2 terminus or both the 3xe2x80x2 and 5xe2x80x2 terminus of the oligonucleotide. Particularly preferred are 2xe2x80x2-O-alkylamines such as 2xe2x80x2-O-ethylamine and 2xe2x80x2-O-propylamine.
Further oligonucleotides of the invention comprise oligonucleotides made up of a plurality of linked nucleotides at least two of which comprise nucleotides that are not 2xe2x80x2-deoxy-erythro-pentofuranosyl nucleotides and that have a C2xe2x80x2 endo type pucker or an O4xe2x80x2 endo type pucker and that are joined together in a contiguous sequence and other nucleotides comprising nucleotides that have a C3xe2x80x2 endo type pucker. Preferred are oligonucleotides having the C3xe2x80x2 endo type pucker nucleotides joined together in a contiguous sequence that is positioned 3xe2x80x2 to the contiguous sequence of the nucleotides having the C2xe2x80x2 endo type pucker or O4xe2x80x2 endo type pucker. Further preferred oligonucleotides are oligonucleotides wherein the nucleotides having the C3xe2x80x2 endo type pucker are joined together in a contiguous sequence that is positioned 5xe2x80x2 to the contiguous sequence of having the C2xe2x80x2 endo type pucker or O4xe2x80x2 endo type pucker. Additional preferred oligonucleotide are oligonucleotides where a portion of the nucleotides having the C3xe2x80x2 endo type pucker are joined together in a contiguous sequence that is positioned 3xe2x80x2 to the contiguous sequence of nucleotides having the C2xe2x80x2 endo type pucker or O4xe2x80x2 endo type pucker and a further portion of nucleotides having the C3xe2x80x2 endo type pucker are joined together in a contiguous sequence that is positioned 5xe2x80x2 to the contiguous sequence of nucleotides having the C2xe2x80x2 endo type pucker or O4xe2x80x2 endo type pucker.